Naked digger and cancer: who's who?

Olga Volkova, "Biomolecule"

The first half of 2016 brought two interesting news about naked diggers – as usual, bad and good. It turned out that malignant neoplasms occasionally, but still affect these long-lived rodents. That, however, does not refute the fact of their phenomenal resistance to cancer. Scientists have succeeded in explaining the mechanisms of this stability. It turned out that induced pluripotent stem cells of diggers, unlike mouse and human, do not provoke tumor growth. And just because of two genetic features. Manipulations with a pair of "warring" genes – suppressing and, conversely, stimulating oncogenesis – led biologists to discover a mechanism of cellular aging specific to diggers and to the possibility of overcoming the main problem of human cell therapy.

A small African eusocial animal with a funny name naked digger (English naked mole-rat, Lat. Heterocephalus glaber) became famous due to unprecedented life expectancy for rodents, immunity to cancer and a number of unique physiological and biochemical features. Biomolecule told in detail about the possible reasons for the resilience of these underground animals and their almost equally lucky relatives [1]. Until 2016, not a single case of the development of malignant neoplasms in diggers was observed. But time and meticulousness of biologists have done their job.

Oncopathology in a naked digger: the first case histories

In the February issue of Veterinary Pathology, a report appeared about two confirmed cases of cancer in diggers from American zoos – Brookfield (Illinois) and National (Smithsonian) (Washington) [2]. Tumors of different nature were found in elderly (over 20 years old) male workers.

In the first case, an asymptomatic mobile 1.5-centimeter neoplasm was extracted from the armpit. Based on cytological, histochemical, immunohistochemical and ultrastructural characteristics, the tumor was classified as an undifferentiated adenocarcinoma of unknown origin – from epithelial cells of either mammary or salivary glands, although the variant of metastasis of another tumor was also not excluded. The number of mitoses in the samples generally did not exceed 0-1 in the field of view (at 400x magnification) and only reached 4 in places. Local invasion could not be estimated, since the transformation zone reached the boundaries of the extracted tissue. However, three months after the operation, only normal scarring was observed in the armpit of the animal – no signs of relapse. It was not possible to assess the effect of the tumor on the well-being and life expectancy of the digger: as for before, after the operation, his health and social adaptation did not suffer.

But the second case can hardly be called asymptomatic, although the cancer was detected posthumously: the animal had to be euthanized due to incurable severe dermatitis and progressive weight loss. It is possible that neuroendocrine carcinoma (obsolete name – carcinoid) detected during the autopsy of the stomach could provoke similar symptoms. Such a tumor occurs from enterochromaffin-like cells secreting histamine. In the euthanized digger, cancer cells penetrated into the mucous and submucosal membranes of the stomach wall, in places – into the muscle layer and even into the adjacent thin-walled vessels (apparently lymphatic), however, they differed in low mitotic activity and did not form metastases.

Previously, hyperplastic changes had already been recorded four times in the same colony of diggers at the Brookfield Zoo. All the findings were accidental: tissue overgrowth was detected during the autopsy of individuals euthanized for other reasons (these animals, for example, can be seriously injured in family skirmishes). Unlike those apparently benign formations, two new cases of neoplasia left no doubt that naked diggers can develop cancer. Another question is that this happens phenomenally rarely. And it would be strange if long–term and purposeful observations would never reveal cancer in this species - these are not some kind of hydra, but real mammals. It is impossible to exclude a special predisposition of specific colonies to neoplasia: after all, single individuals participate in reproduction, and it is likely that some rare mutations will be fixed in the population.

The authors of the article [2] draw readers' attention to the fact that the identified cases in no way refute the fact of the outstanding immunity of naked diggers to cancer. But a thorough analysis of these cases is necessary to develop criteria for the diagnosis of benign and malignant tumors and precancerous conditions in this animal species, which is increasingly being used as a model by biogerontologists and oncologists. And they use it, by the way, successfully.

Useful "settings" of the digger: stem cells with a surprise

Earlier, a group of researchers from the USA showed that the extremely high-molecular hyaluronic acid specific for this species may be related to the anti-cancer protection of diggers, but not just it, but the features of "hyaluronic" signaling, and in particular its final stage – the induction of the INK4 genetic locus. This locus encodes the tumor suppressors p15INK4b, Arf and p16INK4a in a number of animals (and humans too), and the naked digger also has a unique additional product – a hybrid of p15INK4b and p16INK4a [1, 3], called pALTINK4a/b. The synthesis of the latter is induced under various stressful influences and in the process of early contact inhibition of cell division, which is why digger fibroblasts in culture are cautious, stopping division at the slightest contact with neighbors and thereby preventing tumor formation. It is not surprising that the INK4 locus (aka CDKN2A) is very often mutated in cancer in humans, and in addition, is involved in the development of certain types of aging [1].

And this locus again attracted the attention of biologists, but this time Japanese. The main person involved in the new case was his other product, Arf (alternate reading frame protein), which came out in a rather unexpected way – when manipulating induced pluripotent stem cells (iPSCs) of a digger [4].

As biomolecule has repeatedly said, the production of such cells, that is, the reprogramming of somatic cells into dedifferentiated, resembling embryonic stem cells, can be extremely useful both for the treatment of many pathologies and for research purposes [5-7]. However, such reprogramming requires the temporary expression of an "oncogenic cocktail" – the Oct3/4, Sox2, Klf4 and c-Myc (OSKM) genes encoding transcription factors. Their activity brings the process of creating iPSCs closer to oncogenesis: gene expression, epigenetic profile and metabolism change synonymously in cells. Like embryonic stem cells, iPSCs, even briefly expressing this "cocktail" in vivo, can provoke the formation of tumors, most often teratomas [8]. Moreover, the process of tumor transformation does not even require any mutations, it is enough to change the profile of DNA methylation [9]. The risk of developing tumors prevents the introduction of cell therapy into clinical practice, therefore, ways to reduce this risk are being sought in completely different directions: in modifying the composition of reprogramming factors, and in forcing the death of iPSCs unable to fully differentiate, and in the internal reserves of these cells [10].

Japanese researchers decided to test whether animal cells resistant to cancer are capable of reprogramming and tumor formation. The skin fibroblasts of the naked digger fit perfectly. It turned out that it is quite possible to obtain iPSCs from them using retroviral vectors that temporarily express a mouse OSKM cocktail. However, oddly enough, these iPSCs, unlike mouse and human ones, did not form teratomas after transplantation into the testicles of immunodeficient mice (Fig. 1).

Figure 1. Testicles of immunodeficient mice or their tumors after IPSC transplantation of different origin. From top to bottom: a human (after 10 weeks), a mouse (after 4 weeks) and a naked digger (after 10 and 20 weeks). Figure from [4].

To find out which factors are responsible for the oncogenicity of stem cells, and which ones prevent it in digger iPSCs, scientists analyzed the transcription profile of cells (using RNA sequencing [11] and quantitative real-time PCR). As a result, two significant features of the digger's IPSC were revealed.

Active long–term synthesis of one of the products of the INK4 – Arf locus (aka - p19ARF in mice and p14ARF in humans), which helps to prevent excessive cell division: it stabilizes the cell cycle controller p53 by blocking the Mdm2 enzyme that destroys it. As is known, p53 acts through the activation of p21 synthesis (this protein participates in cell differentiation and is able to inhibit their division even without the "order" of p53) – and as if to confirm, the digger's iPSCs showed unusual activity of its gene. In human and mouse cells, the synthesis of INK4 locus products stopped already at the early stages of reprogramming. Moreover, another study showed that the Arf and p16INK4a proteins in the digger are shorter than in humans and mice, and they reliably block the fibroblast cell cycle in response to stress [12].

The "silence" of the ERas gene, an oncogenicity factor of mouse stem cells acting through activation of the PI3K/AKT pathway and belonging to the infamous Ras family. As it turned out, this silence is not due to some regulatory moments, but to a banal mutation that introduced a premature stop codon into the digger's ERas gene, which means that it determined the synthesis of an inferior "pest protein".

Interestingly, both features are directly related to the signaling pathways selected as possible targets for life-prolonging therapy [13]*.

* – The article [13] presents a scheme where the described signaling pathways meet with the well-known enzyme mTOR, whose active work, according to modern concepts, shortens life.

But in order to confirm the involvement of both findings in the amazing cancer resistance of the digger's IPSC, it was necessary to silence their Arf gene and compensate for the defective ERas with workers, mice. This led to suppression of p21 synthesis and induction of the AKT pathway, respectively [4]. A special test for growth in soft agar and transplantation of such modified iPSCs into the testicles of mice showed that switching off Arf stimulates oncogenesis more strongly than activation of ERas, and together these changes lead to the formation of large teratomas.

On the other hand, stable expression of the Arf transgene in mouse iPSCs significantly protected them from tumor transformation, however, in the same teratomas formed, the work of this gene was suppressed.

Well, the most interesting thing happened when researchers studied the reaction of digger fibroblasts to Arf shutdown during IPSC production. In mice, this manipulation increased the efficiency of reprogramming – similar to the knockdown of the Ink4a sequence encoding p16. But the digger's fibroblasts not only refused to divide and rejuvenate, but acquired all the signs of a senescent status. Aging was also observed when Arf was turned off in stressed fibroblasts in which this gene was derepressed in response to activation of the c-Myc oncogene or serial cell passages. Arf and Ink4a derepression is a normal reaction of fibroblasts of many mammals to stresses: reprogramming, activation of oncogenes, replicative stress. Stressed cells age – and this is their first line of defense against oncogenesis. The digger, apparently, formed an additional line, triggered in the case of suppression of Arf synthesis in stressed cells (Fig. 2). This phenomenon researchers called aging induced by Arf suppression (ARF suppression-induced senescence, ASIS).

Figure 2. Reprogramming of fibroblasts of a naked digger and a mouse. The features of the Arf gene and the mutation of the ERas gene (indicated by crossing out) "turn off" the oncogenic potential of the digger's IPSC. The digger's genes are indicated in capital letters. OSKM is a "genetic cocktail" for reprogramming (Oct4, Sox2, Klf4, c-Myc). Senescence of cells is meant by aging. mERas is a mouse, that is, a full-fledged variant of the gene. RIS is aging induced by reprogramming. ASIS is aging induced by Arf suppression. Figure from [4], adapted.

Japanese biologists managed to penetrate a little into the mystery of the work of this unique mechanism. They studied the activity of a number of cell cycle inhibitors in digger fibroblasts, which usually regulate cell aging. It was previously shown that aging of mouse fibroblasts was induced by hypophosphorylation of Rb protein (only phosphorylated Rb allows the cell to prepare for division) and phosphorylation of ACT. Hypophosphorylation of Rb – a key, like p53, controller of cell proliferation – is usually caused by the work of such cycle inhibitors as p27, p21 and p16. However, in digger fibroblasts with ASIS, these inhibitors were not involved in hypophosphorylation of Rb. Phosphorylation of the ACT occurred together with the activation of the proliferative ERK pathway.

Thus, the resistance of the naked digger IPSC to oncogenesis, according to the authors of the work [4], is due to the specific regulation of the Arf gene and damage to the ERas gene. Further study of the ASIS phenomenon will help to fully understand the causes of cancer resistance of this animal and, probably, will contribute to improving the safety of human iPSCs.

Literature

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Portal "Eternal youth" http://vechnayamolodost.ru  18.07.2016